Cytoplasmic calcium and the membrane-associated cytoskeleton of the microvillus and basolateral membranes are important for the efficient absorption of nutrients by the intestine epithelium. The cytoskeleton stabilizes the microvillus surface area y linking the membrane via a llOK-calmodulin complex to an underlying fibrillar bundle composed of actin filaments crosslinked by villin. However. in vitro studies how that in high calcium (greater than MuM). villin fragments actin filaments into short pieces and the unsupported membrane vesiculate. Our studies show that villin is a modular protein composed of separate actin-binding domains and regulated by calcium and phospholipid. The complete villin sequence does not display homology with calmodulin-like calcium- binding sites and together with the phospholipid-binding activity suggests villin is a member of a newly recognized class of calcium- binding proteins. Our studies have presented a framework for studying the relationship between calcium and phospholipid-binding to villin domains and the activation of actin bundling and severing activity. Our goals in this five year renewal are the following: 1. to map the actin, villin, and calcium binding sites by antibody- end-labelling, chemical crosslinking, protein sequencing, protein footprinting, and chemical modification techniques. 2. to synthesize peptides that display specific actin- and calcium- binding activity. 3. to crystallize complexes formed between villin domains and actin. 4. to express the villin cDNA and test the function of different domains by deletion analysis of the expressed protein. 5. to start new studies on cytoskeletal proteins (the llOK- calmodulin complex) that bind actin to integral membrane proteins. The molecular details of calcium-cytoskeleton interactions with the membrane have important health-related significance because loss of microvillar membrane surface area and impaired ion transport are prominent defects that characterize intestinal malabsorption disorders.